A challenge in developmental neuroscience is to understand the molecular mechanisms underlying diversification of neuronal phenotypes derived from multipotent progenitor cells. The neural crest is a population of multipotent progenitors that gives rise to a rise to a diverse array of cell types including neurons of the peripheral nervous system. The differentiation of neural crest-derived cells into sympathetic catecholaminergic (CA) neurons requires exposure to growth factors encountered during their migration from the neural tube and at sites of localization. The mechanisms that control the generation of specific cell types are largely unknown for neural crest-derived cells, but most likely involve differential patterns of gene expression in response to both intrinsic and extrinsic cues. Neurogenic basic helix-loop-helix (bHLH) DNA binding proteins have been shown to control cell fate and phenotypic expression. However, there is little detailed information about the identity or actions of bHLH factors during vertebrate neurogenesis. Our hypothesis is that the expression of dHAND and eHAND, two novel bHLH factors we have recently cloned from chicken, is linked via growth factors to the regulation of neurogenesis and specification of CA neurotransmitter phenotype in neural crest- derived cells. The project will utilize molecular, cell biological and immunological approaches to define the role the dHAND and eHAND play in the development of neural crest-derived sympathetic ganglia. First, in situ hybridization combined with immunocytochemistry will be used to identify cell types expressing transcripts encoding dHAND and eHAND. The spatial and temporal patterns of expression of HAND gene transcripts will be determined. Second, to determine the functional role of dHAND and eHAND in the differentiation of CA neurons, the consequences of over expression of HAND gene products in neural crest-derived cells will be studied both in vitro and in vivo. The consequences of ectopic expression of HAND genes will be determined. Third, to identify upstream regulators and downstream targets of HAND proteins we will determine if growth factors known to influence CA differentiation affect HAND gene expression. Interactions between HAND proteins and other transcription factors important in CA differentiation will be assessed. Finally, we will try to rescue CA differentiation of crest cells from MASH1 null mice by expressing HAND proteins. Delineating how neural crest-derived cells begin to exhibit cell type-specific gene expression will enhance our understanding of basic developmental mechanisms underlying neurogenesis, and the origins of clinical manifestations of crest-derived neuroectodermal tumors.